The first therapeutic use of corticosteroids was demonstrated in the 1950's with the introduction of cortisone acetate treatment for rheumatoid arthritis. Further studies demonstrated that the insertion of unsaturation into the 1,2 position of hydrocortisone and cortisone caused the resultant steroids, prednisolone and prednisone, to have enhanced potency and to cause less drug-induced salt retention. Subsequently, most other steroids used for the treatment of corticoid-responsive diseases have been synthesized so that they contain a double bond in the 1,2 position of the steroid molecule. In 1977, two U.S. patents were issued which represent new approaches to the synthesis of corticosteroids from sterol precursors. U.S. Pat. No. 4,035,236 covers a process for preparing 9.alpha.-hydroxyandrostenedione via fermentation of sitosterol, stigmasterol, or cholesterol. U.S. Pat. No. 4,041,055 discloses a general process for the synthesis of medically useful corticosteroids from this androstene. Intermediates covered in this chemistry can possess a 3-keto-.DELTA..sup.4,.sup.9(11) configuration.
Several methods have been described in the literature for microbiological introduction of a 1,2-unsaturated bond in the A-ring of steroid compounds that are important intermediates in the synthesis of medically useful steroids. U.S. Pat. No. 2,837,464 describes the 1-dehydrogenation of steroids by the addition of steroid substrate to fermentation broths of Arthrobacter simplex. However, the total utility of this process is limited. This bacterium and other 1-dehydrogenating microorganisms can further degrade certain steroid molecules resulting in a lower final yield as well as undesired side products.
U.S. Pat. No. 3,091,575 discloses an improved method for steroid 1-dehydrogenation by the intermixing of the steroid, an electron carrier, and bacterial cells that have been pretreated with a lower alkanol or lower alkanone such as acetone. This pretreatment reduces one of the undesirable activities, 20-keto reductase activity, in the cells.
U.S. Pat. No. 3,047,469 discloses a different type of a process which comprises subjecting a steroid which is saturated in the 1,2 position to a mixture of an electron carrier and a steroid-1-dehydrogenase containing extract from a microorganism selected from the group consisting of Nocardia, Corynebacterium, Mycobacterium and Cylindrocarpon. This method overcomes several disadvantages encountered by the use of living organisms, including the reduction of accompanying side reactions that lead to steroid degradation.
U.S. Pat. No. 3,091,575 documents a process to eliminate the destruction of the desired product by the addition of inhibitors, such as quinonoid type compounds, to the fermentation broth prior to or at the same time as the addition of the steroid substrate.
The utility of exogenous electron carriers is sometimes limited by the toxic effects that they may exert on the enzyme system (J. H. Quasiel. Methods in Enzymology. S. P. Colowick and N. O. Kaplan, Eds, Academic Press, Inc., New York, Vol. 4, pp. 329-336, 1957). Yang and Studebaker (Biotechnology and Bioengineering, 20, pp. 17-25, 1978) discussed the potential toxicity of the electron carrier phenazinemethosulfate (PMS) on the steroid-1-dehydrogenase of Pseudomonas testosteroni caused by superoxide and peroxide formation. However, they concluded that the 1-dehydrogenase activity was essentially unaffected by the presence of PMS. They suggested that this strict aerobe possessed sufficient superoxide dismutase and catalase activities to remove any superoxide and peroxide that was formed before damage to the steroid 1-dehydrogenase occurred.
The subject invention process represents an improved steroid 1-dehydrogenation bioconversion in the presence of added electron carriers that is not suggested or disclosed by the prior art.